Abstract Adhesion of cells to extracellular matrices is a fundamental characteristic of all multicellular organisms. Adhesion provides not only structural links between the intracellular cytoskeleton and the extracellular environment, but also provides sites of signal transduction that affect many aspects of cell behavior. This grant is aimed at understanding how signals from cell-matrix adhesions regulate members of the Rho family of GTPases, which are themselves key regulators of the cytoskeleton and many intracellular processes. The goal of the first aim is to determine how adhesion to the extracellular matrix protein fibronectin stimulates RhoA. Having recently identified guanine nucleotide exchange factors, such as Lsc, that are involved in this process, we aim to determine the signaling pathways that lead to their activation. Our finding that adhesion stimulates Lsc tyrosine phosphorylation and promotes Lsc binding to vinculin suggests possible mechanisms of regulation and will be investigated further. We will also examine how mechanical tension and substrate rigidity/pliability regulate RhoA activity. Here we have found that some but not all of the same exchange factors are involved. We will investigate the signaling pathways downstream from mechanical tension that lead to exchange factor activation. In the second aim, we will follow up the discovery that reactive oxygen species can activate Rho proteins by transient oxidation of cysteine sulfhydryls without involvement of exchange factors. Using redox-resistant mutants of Rho GTPases, we will determine the role of Rho protein activation by cysteine oxidation in events such as adhesion and in response to growth factor stimulation. In the final aim, we will focus on the Rho GTPase sequestering protein RhoGDI, which we have recently found contributes to crosstalk between different Rho family members. Competitive interactions of Rho proteins for binding to RhoGDI will be explored. We will investigate how Rho proteins are released from RhoGDI in response to adhesion and determine the pathways that lead to their degradation following displacement from RhoGDI. Finally, we will explore the role of RhoGDI in mediating the trafficking of Rho proteins from their sites of postranslational modification to their sites of action at the plasma membrane.